1,4-diazabicyclo [2.2.2] oct-2-ylmethyl derivatives, their preparation and therapeutic application

ABSTRACT

PCT No. PCT/FR97/02174 Sec. 371 Date Jun. 4, 1999 Sec. 102(e) Date Jun. 4, 1999 PCT Filed Dec. 2, 1997 PCT Pub. No. WO98/24790 PCT Pub. Date Jun. 11, 1998The invention concerns compounds of general formula (I) in which R1 represents a methyl group, X1 represents a hydrogen atom, or OR1 and X1 together form a group of formula -O(CH2)2 -,-O(CH2)3-, -O(CH2)2O- or -O(CH2)3O-, X2 represents a hydrogen atom or an amino group, and X3 representing a halogen atom. The compounds are ligands of serotoninergic receptors of the 5-HT3 and/or 5-HT4 types, and act as 5-HT4 agonists and/or 5-HT3 antogonists.

The subject-matter of the present invention is1,4-diazabicyclo[2.2.2]oct-2-ylmethyl benzoate derivatives, theirpreparation and their application in therapeutics.

The compounds of the invention correspond to the general formula (I)##STR2## in which R₁ represents a methyl croup,

X₁ represents a hydrogen atom,

or

OR₁ and X₁ together form a group of formula --O(CH₂)₂ --, --O(CH₂)₃ --,--O(CH₂)₂ O-- or --O(CH₂)₃ O--,

X₂ represents a hydrogen atom or an amino group, and

X₃ represents a halogen atom.

They can exist in the form of free bases or of addition salts withacids. Furthermore, they comprise an asymmetric carbon atom in thediazabicyclooctane ring and can therefore exist in the form of pureenantiomers or of mixtures of enantiomers.

According to the invention, the compounds of general formula (I) areprepared by a process illustrated by the following scheme. ##STR3##

Ethyl 1,4-diazabicyclo[2.2.2]octane-2-carboxylate of formula (III)(described in Helv. Chimica Acta (1962), Vol. XLV, Part VII, No. 2732383) is first prepared by reaction between piperazine and ethyl2,3-dibromopropanoate in an inert solvent, for example toluene orbenzene, and optionally in the presence of an organic base, for exampletriethylamine, then the ester obtained is reduced to1,4-diazabicyclo[2.2.2]octane-2-methanol of formula (IV) (described inKhim. Farm. Zh. (1989), 23, 30-35 and in Khim. Geterosikl. Soedin.(1980), 10, 1404-1407), for example by means of lithium aluminiumhydride, and, finally, the alcohol obtained is treated with a benzoicacid derivative of general formula (V), in which R₁, X₁, X₂ and X3 areas defined above, according to any known esterification method, forexample by activation of the acid by means of an imidazolide and bycoupling of the latter with the alcohol, converted beforehand toalkoxide, for example by means of butyllithium.

Piperazine and ethyl 2,3-dibromopropanoate are commercially available.Some benzoic acid derivatives of general formula (V) are commerciallyavailable; the others can be prepared by methods such as those disclosedin J. Med. Chem. (1993), 36, 4121-4123 and in Patent ApplicationsEP-0234872, WO-9305038 and ES-2019042 or by saponification ofcorresponding esters, such as those disclosed in Patents DE-3001328 andDE-36433103.

The following example illustrates in detail the preparation of acompound according to the invention. The elemental microanalyses and theIR and NMR spectra confirm the structures of the compounds obtained.

EXAMPLE (1,4-Diazabicyclo[2.2.2]oct-2-yl)methyl8-amino-7-chloro-2,3-dihydro-1,4-benzodioxine-5-carboxylate.

1. Ethyl 1,4-diazabicyclo[2.2.2]octane-2-carboxylate.

16.9 ml (116 mmol) of ethyl 2,3-dibromopropanoate, in solution in 250 mlof toluene, 10. g (116 mmol) of piperazine and 49.ml (348 mmol) oftriethylamine, in solution in 150 ml of toluene, are successivelyintroduced into a round-bottomed flask and the mixture is heated at 80°C. with magnetic stirring overnight. The mixture is cooled, thetriethylamine hydrobromide is separated off by filtration, they beingrinsed with ethyl acetate, the solvents are evaporated from the filtrateunder reduced pressure and the residue is distilled at ambient pressure.6.91 g of compound are obtained, which compound is used as is in thefollowing stage.

2. 1,4-Diazabicyclo[2.2.2]octane-2-methanol.

0.21 g (5.43 mmol) of lithium aluminium hydride, in suspension indiethyl ether, is introduced into a round-bottomed flask, the suspensionis cooled to 0° C. using an ice-cold bath, 1 g (5.43 mmol) of ethyl1,4-diazabicyclo[2.2.2]octane-2-carboxy-ate, in solution in 10 ml ofdiethyl ether, is slowly added with magnetic stirring and the mixture isstirred at room temperature for 1 h.

The excess hydride is hydrolysed by slow addition of 0.2 ml of water,0.2 ml of 15% aqueous sodium hydroxide solution and then a further 0.6ml of water, the solid is separated off by filtration, it being rinsedwith chloroform, and the filtrate is evaporated under reduced pressure.

After distillation, 0.43 g of oily product is obtained.

3. (1,4-Diazabicyclo[2.2.2]oct-2-yl)methyl8-amino-7-chloro-2,3-dihydro-1,4-benzodioxine-5-carboxylate.

0.69 g (3.03 mmol) of8-amino-7-chloro-2,3-dihydro-1,4-benzodioxine-5-carboxylic acid, insuspension in 5 ml of anhydrous tetrahydrofuran, is introduced into around-bottomed flask, 0.49 g (3.03 mmol) of N,N'-carbonyldiimidazole isadded, under a nitrogen atmosphere and at room temperature, and themixture is stirred for 2 h. 0.43 g (3.02 mmol) of1,4-diazabicyclo[2.2.2]octane-2-methanol, in solution in 20 ml oftetrahydrofuran, is introduced into another round-bottomed flask and1.21 ml (3.02 mmol) of a 2.5M solution of butyllithium in hexane areslowly added, under an argon atmosphere and with magnetic stirring, andthe stirring is continued for 1 h.

The acid solution prepared above is then slowly added, using a syringe,to the butyllithium solution and stirring is continued for 1 h 10 min atroom temperature.

The solvent is evaporated under reduced pressure, the residue is takenup in ethyl acetate and water, and the organic phase is separated offand washed several times with water. After drying, evaporating, takingup the residue in diethyl ether and recrystallizing from ethanol, 0.35 gof white solid is isolated. Melting point: 217° C.

The chemical structures and the physical properties of some compounds ofthe invention are illustrated in the following table.

                  TABLE                                                           ______________________________________                                                                       (I)                                             ##STR4##                                                                                                              M.p.                                 No.   OR.sub.1 X.sub.1                                                                             X.sub.2                                                                              X.sub.3                                                                            Salt    (° C.)                        ______________________________________                                        1     --OCH.sub.3                                                                            H     H      Cl   --      121-124                              2     --OCH.sub.3                                                                            H     --NH.sub.2                                                                           Cl   --      168-171                              3     --O(CH.sub.2).sub.2--                                                                    H        Cl   HCl (1:2)                                                                             247-250                                4     --C(CH2).sub.2 O--                                                                       --NH.sub.2                                                                             Cl   --      217                                    5     --O(CH2).sub.3 O--                                                                       --NH.sub.2                                                                             Cl   HCl (1:2)                                                                             191-198                                ______________________________________                                         In the "Salt" column, "--" denotes a compound in the baseform and "2HCl"      denotes a dihydrochloride.                                               

The compounds of the invention have formed the subject of tests whichhave demonstrated their advantage as substances possessing therapeuticactivities.

Thus, their affinity for 5-HT₃ serotoninergic receptors was demonstratedby the displacement of the binding of a specific labelled ligand, [³H]-(S)-zacopride.

The study is carried out in vitro on the 5-HT₃ receptors of the ratcortex, essentially as described by Barnes N. M. et al., J. Pharm.Pharmacol. (1988), 40, 548-551. Male Sprague-Dawley rats (OFA, IffaCredo, Lyons, France), with a weight of 200 to 250 g, are humanelykilled and the brain is removed. The cortex is subsequently dissectedand homogenized using a Polytron™ mill (position 7, 20 s) in 20 volumesof Tris buffer (25 mM, ph=7.4 at 22° C.), the homogenate is centrifugedat 45,000 g for 10 min using a Sorvall™ centrifuge equipped with an SS34rotor, and the pellet is resuspended in 10 volumes of Tris buffer andincubated at 37° C. for 10 min with stirring. The suspension is dilutedto 20 volumes using Tris buffer and again centrifuged under the sameconditions, and then the pellet is resuspended in 5 volumes of Trisbuffer and divided into 5 ml aliquot fractions which are frozen at -80°C.

On the day of the experiment, the preparation is thawed at +4° C. andthen diluted 1.2 times using Tris-NaCl incubation buffer (Tris 25 mM,NaCl 150 mM, pH=7.4 at 22° C.).

The membrane suspension (100 μl, 1 mg of proteins) is then incubated at25° C. for 25 min in the presence of 0.5 nM of [³ H]-(S)-zacopride(specific activity 75-85 Ci/mmol, Amersham, Little Chalfont, GreatBritain) in a final volume of 500 μl of Tris-NaCl buffer in the presenceor in the absence of test compound.

The incubation is halted by filtration through Whatman GF/B filterspretreated with 0.1% polyethyleneimine. Each reaction tube is predilutedwith 4 ml of Tris-NaCl buffer and then rinsed three times with 4.5 ml ofof Tris-NaCl buffer.

The filters are cut up beforehand prior to drying in an oven (120° C., 5min). The radioactivity retained on the filters is determined by liquidscintigraphy. The non-specific binding is determined in the presence of10 μM of MDL 72222 (ligand described in the cited article). For eachconcentration of studied compound, the percentage of inhibition of thespecific binding of [³ H]-(S)-zacopride and then the IC₅₀ concentration,the concentration of this compound which inhibits 50% of this specificbinding, are determined. The IC₅₀ values of the compounds of theinvention lie between 0.1 and 0.5 μm.

The compounds of the invention were also studied for their affinity withrespect to 5-HT₄ receptors in the striatum of guinea pigs according tothe method described by Grossman et al. in Br. J. Pharmacol., (1993)109, 618-624. Guinea pigs (Hartley, Charles River, France) weighing 300to 400 g are humanely killed, the brains are removed and the striata areexcised and frozen at -80° C.

On the day of the experiment, the tissue is thawed at +4° C. in 33volumes of HEPES-NaOH buffer (50 mM, pH=7.4 at 20° C.) and ishomogenized using a Polytron® mill, the homogenate is centrifuged at48,000 g for 10 min, the pellet is recovered, resuspended andrecentrifigued under the same conditions and the final pellet isresuspended in HEPES-NaOH buffer, in the proportion of 30 mg of tissueper ml. 100 μl of this membrane suspension are incubated at 0° C. for120 min in the presence of [³ H]GR113808 (ligand described in the citedarticle, specific activity 80-85 Ci/mmol) in a final volume of 1 ml ofHEPES-NaOH buffer (50 mM, pH=7.4), in the presence or in the absence oftest compound. Incubation is halted by filtration through a Whatman GF/Bfilter pretreated with 0.1% polyethyleneimine, each tube is rinsed with4 ml of buffer at 0° C., filtration is again carried out and theradioactivity retained on the filter is measured by liquid scintigraphy.

The non-specific binding is determined in the presence of 30 μMserotonin. The specific binding represents 90% of the totalradioactivity recovered on the filter. For each concentration of studiedcompound, the percentage of inhibition of the specific binding of [³H]GR113808 and then the IC₅₀, the concentration of the test compoundwhich inhibits 50% of the specific binding, are determined. The IC₅₀values of the compounds of the invention lie between 0.015 and 5 μM.

The compounds of the invention were also studied as regards theiragonist or antagonist effects with respect to 5-HT₄ receptors in the ratoesophagus, according to the method described by Baxter et al. in NaunynSchmied. Arch. Pharmacol., (1991) 343, 439. Male Sprague-Dawley ratsweighing from 300 to 450 g are used. An approximately 1.5 cm fragment isquickly removed from the end part of the oesophagus, the muscular layeris removed and the internal muscular mucosal tunic is openedlongitudinally, mounted in an isolated organ vessel containing aKrebs-Henseleit solution at 32° C. oxygenated by a carbogen stream (95%O2 and 5% CO₂) and connected to an isometric transducer under a basaltension of 0.5 g. A contraction of the tissue is induced by the additionof 05 μM of carbachol, there is a wait while the contraction becomesstabilized (15 min), and then the preparation is exposed to serotonin (1μM) in order to quantify the maximum relaxation. The tissue is washedand, after a period of 20 min, 0.5 μM of carbachol is again added andthe preparation is exposed to the study compound, in increasing additiveconcentrations from 0.1 to 1 μM. The compounds which induce a relaxationare characterized as 5-HT₄ agonists. For the compounds which do notinduce relaxation, the preparation is exposed to serotonin in increasingadditive concentrations, from 0.1 nM to a concentration inducing amaximum relaxation, and the relaxation curve due to serotonin, in thepresence of the study compound, is then compared with a control curveprepared in the absence of the said compound. If its presence induces ashift of the curve towards the right, the study compound ischaracterized as a 5-HT₄ antagonist.

Finally, the compounds of the invention were studied as regards theirantagonist effects with respect to 5-HT₃ receptors of the descendingcolon smooth muscle isolated from the guinea pig, according to themethod described by Grossman et al. in Br. J. Pharmacol. (1989), 97,451.

Serotonin (0.1-100 μM), after blocking the receptors of 5-HT₁ and 5-HT₂types (0.1 μM methysergide) and desensitizing the 5-HT₄ receptors (10 μM5-methoxytryptamine), causes a contraction, dependent on theconcentration, of the smooth muscular part of the descending colon ofthe guinea pig, by stimulation of the 5-HT₃ receptors. The contractionsare recorded by isometry.

The antagonist effect of a compound on the 5-HT₃ serotoninergicreceptors is quantified by the measurement of the shift of a controlconcentration-effect curve of serotonin (non-additive increasingsuccessive concentrations), at concentrations of the compound of between1 nM and 0.1 μM, with an incubation of 30 min.

The results of the biological tests carried out on the compounds of theinvention show that they are ligands for serotoninergic receptors of5-HT₃ and/or 5-HT₄ types and that they act as 5-HT₄ agonists orantagonists and/or as 5-HT₃ antagonists.

The compounds can therefore be used for the treatment and prevention ofdisorders in which the 5-HT₃ and/or 5-HT₄ receptors are involved,whether at -he level of the central nervous system, of thegastrointestinal system, of the cardiovascular system or of the urinarysystem.

At the level of the central nervous system, these disorders and problemscomprise in particular neurological and psychiatric disorders such ascognitive disorders, psychoses, compulsive and obsessional behavioursand states of depression and of anxiety. The cognitive disorderscomprise, for example, memory and attention deficits, states of dementia(senile dementias of the Alzheimer's disease type or dementias relatedto age), cerebrovascular deficiencies or Parkinson's disease. Thepsychoses comprise, for example, paranoia, schizophrenia, mania andautism. The compulsive and obsessional behaviours comprise, for example,eating disorders of the loss of appetite or bulimia type. The states ofdepression and of anxiety comprise, for example, anxieties ofanticipatory type (before a surgical operation, before dental treatment,and the like), the anxiety caused by dependence on or withdrawal fromalcohol or drugs, man,-a, seasonal affective disorders, migraines ornausea.

At the level of the gastrointestinal system, these disorders andproblems comprise in particular vomiting induced by an antitumourtreatment, direct or indirect disorders of gastromotility of theoesophagus, of the stomach or of the intestines, or specific complaints,such as dyspepsia, ulcer, gastro-oesophagal reflux, flatulence,irritable bowel syndrome, disorders of intestinal secretion ordiarrhoeas, for example those induced by cholera or by carcinoidsyndrome.

At the level of the cardiovascular system, these disorders and problemscomprise in particular pathologies related, directly or indirectly, tocardiac arrhythmias.

At the level of the urinary system, these disorders and problemscomprise in particular incontinences of all kinds, as well as theircauses or consequences, for example infections, stones or renal damage.

The compounds of the invention can be presented in all forms ofcompositions appropriate for enteral or parenteral administration, suchas tablets, dragees, capsules, including hard gelatin capsules,suspensions or solutions to be swallowed or injected, such as syrups orphials, and the like, in combination with suitable excipients, and indoses which make possible a daily administration of 0.005 to 20 mg/kg.

We claim:
 1. A compound in the form of a pure enantiomer or a mixture ofenantiuomers, having the formula (I): ##STR5## wherein R₁ represents amethyl group, andX₁ represents a hydrogen atom,or OR₁ and X₁ togetherform a group of formula --O(CH₂)₂ --, --O(CH₂)₃ --, --O(CH₂)₂ O-- or--O(CH₂)₃ O--, X₂ represents a hydrogen atom or an amino group, and X₃represents a halogen atom, in the form of the free base or of anaddition salt with an acid.
 2. A process for the preparation ofcompounds according to claim 1, comprising:reacting piperazine and ethyl2,3-dibromopropanoate to form ethyl1,4-diazabicyclo[2.2.2]octane-2-carboxylate; reducing ethyl1,4-diazabicyclo[2.2.2]octane-2-carboxylate to form1,4-diazabicyclo[2.2.2]octane-2-methanol; reacting1,4-diazabicyclo[2.2.2]octane-2-methanol with a benzoic acid derivativeof formula (V): ##STR6## wherein R₁, X₁, X₂ and X₃ are as defined inclaim 1; and optionally forming an addition salt with an acid.
 3. Apharmaceutical composition, comprising a pharmaceutically effectiveamount of at least one compound according to claim 1, in combinationwith a pharmaceutically acceptable excipient.
 4. A pharmaceuticalcomposition according to claim 3, wherein the composition is in the formof a tablet, dragees, capsule, hard gelatin capsule, suspension orsolution to be swallowed or injected, syrup, or phial.
 5. A method oftreating or preventing a serotonin-related disorder, comprisingadministering a pharmaceutically effective amount of at least onecompound according to claim 1 to a patient in need thereof.
 6. Themethod of claim 5, wherein said serotonin-related disorder is related tothe 5-HT₃ receptors, 5-HT₄ receptors, or both.
 7. The method of claim 5,wherein said disorder is involved at the level of the central nervoussystem, gastrointestinal system, cardiovascular system, or urinarysystem.
 8. The method of claim 7, wherein said central nervous systemdisorder is selected from the group consisting of cognitive disorders,psychoses, compulsive behaviors, obsessional behaviors, depression, andanxiety.
 9. The method of claim 8, wherein said cognitive disorder isselected from the group consisting of memory and attention deficits,states of dementia, Alzheimer's senile dementias, dementias related toage, cerebrovascular deficiencies, and Parkinson's disease.
 10. Themethod of claim 8, wherein said psychoses are selected from the groupconsisting of paranoia, schizophrenia, mania, and autism.
 11. The methodof claim 8, wherein said compulsive behaviors or obsessional behaviorsare the eating disorders loss of appetite or bulimia.
 12. The method ofclaim 8, wherein said depression or anxiety disorders are selected fromthe group consisting of anticipatory anxiety, anxiety caused bydependence on or withdrawal from alcohol or drugs, mania, seasonalaffective disorders, migraines, and nausea.
 13. The method of claim 7,wherein said gastrointestinal system disorder is either vomiting inducedby an antitumour treatment, or direct or indirect disorders ofgastromotility of the oesophagus, stomach and/or intestines.
 14. Themethod of claim 7, wherein said gastrointestinal system disorder isselected from the group consisting of dyspepsia, ulcer,gastro-oesophagal reflux, flatulence, irritable bowel syndrome,disorders of intestinal secretion, diarrhea, diarrhea induced bycholera, and diarrhea induced by carcinoid syndrome.
 15. The method ofclaim 7, wherein said cardiovascular system disorder is a pathologyrelated to cardiac arrhythmias.
 16. The method of claim 7, wherein saidurinary system disorder is incontinence.
 17. The method of claim 7,wherein said urinary system disorder is a disorder related to the causeor consequence of incontinence, infections, stones, or renal damage.